JP2019502129A - Discontinuous sample fraction integration device and dual on-line multidimensional liquid chromatography system equipped with the same - Google Patents

Abstract

A discontinuous sample fraction integration unit and a dual on-line multifunction liquid chromatography device comprising the same are disclosed. A discontinuous sample fraction integration unit according to an embodiment of the present invention is connected to a sample supply module that supplies a sample to be analyzed; and a sample supply module, and receives a sample continuously from the sample supply module. Set the unit sample supply time that is equally divided into the total sample supply time to be received, set the unit fraction section that equally divides each of the plurality of unit sample supply times into multiple, and then within the unit sample supply time A sample fractionation module for collectively storing samples received during corresponding unit fractionation intervals to obtain a plurality of fraction samples; [Selection] Figure 3

Description

  The present invention relates to a discontinuous sample fraction integration device and a dual on-line multifunction liquid chromatography system equipped with the same, and more particularly, a discontinuous method that reduces sample complexity and increases fraction sample uniformity. The present invention relates to an integrated sample fractionation device and a dual online multifunction liquid chromatography system equipped with the same.

  In proteomics, a study that comprehensively studies all proteins expressed under specific conditions in vivo, a method that combines a liquid chromatography system and mass spectrometry (LC-MS / MS) It is emerging as an important technology.

  Among them, the bottom-up or shotgun proteomics method, in which a protein is hydrolyzed into a small unit peptide and analyzed, is widely used.

Although bottom-up proteomics methods have proven useful, they inevitably cause sample complexity. For example, when a protein expressed from about 20,000 dielectrics is hydrolyzed into peptides, tens of millions or more of peptides can be obtained. In vivo proteins also have a wide concentration dynamic range (10 ¹⁰ ). Therefore, in order to solve such sample complexity and perform effective protein body analysis, a separation method having high resolution is required.

  As a technique for solving such a problem, there is a two-dimensional liquid chromatography technique. This is an integrated use of two different separation methods.

  There are two factors that determine the success or failure of two-dimensional liquid chromatography. The first is the separation efficiency of each separation method, and the second is the separation orthogonality.

  Separation orthogonality means that the sample mixture must be separated by different methods in different ways, i.e. by different physicochemical properties.

  One of the two-dimensional separation methods developed and used in recent years is that the sample is fractionated according to the degree of hydrophobicity under basic pH conditions, and then the separated fractionated sample is subjected to acidic pH conditions. They are separated again according to the degree of hydrophobicity (two-dimensional reverse phase liquid chromatography-reverse phase liquid chromatography, 2D RP-RPLC).

  Basically, separating twice by hydrophobicity cannot be said to have separation orthogonality, but since peptides have different charge distributions depending on their unique amino acid sequences under different pH conditions, they are the same peptide. However, they have different degrees of hydrophobicity. Thus, the 2D RP-RPLC method has separation orthogonality.

  However, the 2D RP-RPLC method cannot have perfect separation orthogonality. FIG. 1 shows that after performing RPLC separation at pH 10, each fraction sample was further subjected to RPLC separation at pH 2.6 and analyzed with a mass spectrometer, and the distribution of the identified peptides was analyzed for the elution time of each separation method. The distribution at is shown. When FIG. 1 is seen, many parts where the peptide does not appear can be seen. This means that the 2D RP-RPLC method could not use all of the separation space for two-dimensional separation.

  A method developed to solve such problems is post-fractionation discontinuous integration technology. Referring to FIG. 2a, the post-fractionation discontinuous integration technique is divided into 96 (1st to 96th) according to the order of elution when performing RPLC separation under basic pH conditions. Furthermore, it integrates into 24 fraction samples. At this time, instead of continuous integration, after preparing No. 1 to No. 24 as 24 fraction samples, No. 25 is further integrated into the first fraction sample and No. 26 to the second fraction sample. Integrate into the sample. In this case, the first fraction sample is Nos. 1, 25, 49 and 73, the second fraction sample is Nos. 2, 26, 50 and 74, and the last 24th fraction sample. Nos. 24, 48, 72, and 96 are integrated. When discontinuous integration is performed in this manner, the entire separation space for two-dimensional separation can be used as shown in FIG. 2b.

  By such a non-continuous integration technique after fractionation, the separation orthogonality of the 2D RP-RPLC technique is fully utilized to ensure separation orthogonality.

  However, researchers need to perform this integration process directly, which requires a lot of human power and time. In addition, in order to conduct an LC-MS / MS experiment under acidic pH conditions, the fraction sample obtained by the off-line method is reduced in volume to a solvent capable of LC-MS / MS experiment. Furthermore, many processing steps such as melting are required, and sample loss may occur during this process, which may affect the reproducibility of the experiment. Therefore, it is necessary to develop a liquid chromatography system in which such a process is automatically performed after sample injection.

M Gilar et al, Anal Chem, 2005, 77, 6426-6434. J. et al. M.M. Park et al, Sci. Rep. 5, 18189; doi: 10.1038 / srep18189 (2015)

  Therefore, the technical problem to be solved by the present invention is to improve fraction reproducibility by automating the discontinuous sample fraction integration process which reduces the complexity of the sample and increases the uniformity of the fractionated sample. And providing a discontinuous sample fraction integration device capable of preventing sample loss and a dual on-line multifunction liquid chromatography system equipped with the same.

  According to one aspect of the present invention, a sample supply module for supplying a sample to be analyzed; and a total sample supply connected to the sample supply module for continuously receiving the sample and receiving the sample from the sample supply module After setting the unit sample supply time in which the time is equally divided into multiple units, and setting the unit fraction section in which each of the plurality of unit sample supply times is equally divided into multiple units, the corresponding unit minutes in each unit sample supply time are set. A discontinuous sample fraction integration device may be provided that includes a sample fractionation module that collectively stores the samples received during fractionation sections to obtain a plurality of fraction samples.

  The sample fractionation module is connected to the sample supply module, and a first fractionation valve through which the sample flows; a second fractionation valve provided adjacent to the first fractionation valve; and one end of the first fractionation module The unit sample is connected to the fractionation valve, the other end is connected to the second fractionation valve, and a plurality of unit sample sections are provided corresponding to the number of the unit fractionation sections within the unit sample supply time. A fraction sample storage loop may be included for sequentially and integrally storing the samples received during the corresponding unit fraction intervals within a supply time.

  The first fraction valve is a fraction sample inlet port through which the sample flows from the sample supply module; provided adjacent to the fraction sample inlet port and connected to one end of the plurality of fraction sample storage loops. A plurality of first fraction sample storage loop connection ports; and one of the fraction sample inflow port and the plurality of first fraction sample storage loop connection ports corresponding to the unit fraction section. A plurality of second fraction sample storage loop connection ports to which the other ends of the plurality of fraction sample storage loops are respectively connected; A fraction sample discharge port provided adjacent to a plurality of second fraction sample storage loop connection ports and connected to the other end of any one of the plurality of fraction sample storage loops to discharge the fraction sample. And a second connection bar for connecting any one of the fraction sample discharge port and the plurality of second fraction sample storage loop connection ports so as to communicate with each other, corresponding to the unit fraction section. it can.

  The sample supply module is connected to a first sample supply valve to which a first pump for supplying a first solvent and a sample injector are connected; and to the first sample supply valve, and the sample is supplied from the first sample supply valve. A second sample supply valve may be included to receive and supply the fraction sample input port.

  The first sample supply valve includes: a first sample inflow port connected to the sample injector; a first sample discharge port provided adjacent to the first sample inflow port; a first connected to the first pump. A solvent inlet port; a first solvent outlet port provided adjacent to the first solvent inlet port and connected to the second sample supply valve; and a first sample storage loop connection to which both ends of the sample storage loop are connected A port and a second sample storage loop connection port, wherein each of the first sample storage loop connection port and the second sample storage loop connection port is connected to the first sample inflow port and the first sample discharge port The sample is stored in the sample storage loop, and each of the first sample storage loop connection port and the second sample storage loop connection port receives the first solvent inflow. Can be supplied to the sample in the second sample supply valve by implanting the first solvent to the sample storage loop in a state of being connected to the over preparative said first solvent discharge port.

  The second sample supply valve is a second sample inlet port connected to the first solvent outlet port; a second sample provided adjacent to the second sample inlet port and connected to the fractionated sample inlet port A discharge port; and a sample separation column that is connected at both ends to the second sample inflow port and the second sample discharge port, and the sample is eluted into the second sample discharge port, and is eluted from the sample separation column. The sample can be supplied to the fraction sample inlet port.

  According to another aspect of the present invention, a sample to be analyzed is continuously received, and a unit sample supply time is set by equally dividing a total sample supply time for receiving the sample into a plurality of unit sample supply times. A plurality of fraction samples obtained by integrating and storing the samples received during the corresponding unit fraction intervals within each unit sample supply time A first solid phase in which a first reverse phase liquid chromatography column and a first reverse phase liquid chromatography column are connected and connected to the first reverse phase liquid chromatography column; A dual column valve with a second solid phase extraction column coupled to an extraction column and the second reverse phase liquid chromatography column; and the discontinuous sample fraction integration device and the duplex The fraction samples provided between the ram valves and sequentially supplied from the discontinuous sample fraction integration device are alternately supplied to the first solid phase extraction column and the first reverse phase liquid chromatography column or the second. A dual online multifunction liquid chromatography system can be provided that includes a solid phase extraction column and a column selection module that feeds the second reversed phase liquid chromatography column.

  The discontinuous sample fraction integration device is connected to the sample supply module for supplying the sample; and the sample supply module, and continuously receives the sample from the sample supply module to obtain a plurality of fraction samples. A sample fractionation module that sequentially supplies the plurality of fractionation samples to the column selection module can be included.

  The sample fractionation module is connected to the sample supply module, and a first fractionation valve through which the sample flows; a second fractionation valve provided adjacent to the first fractionation valve; and one end of the first fractionation module The unit sample is connected to the fractionation valve, the other end is connected to the second fractionation valve, and a plurality of unit sample sections are provided corresponding to the number of the unit fractionation sections within the unit sample supply time. A plurality of fraction sample storage loops may be included for sequentially and integrally storing the samples received during the corresponding unit fraction intervals within a supply time.

  The first fractionation valve is provided adjacent to the first fraction sample inflow port through which the sample flows from the sample supply module; and is provided adjacent to the first fraction sample inflow port. A plurality of first fraction sample storage loop connection ports each connected at one end; and any one of the fraction sample inflow port and the plurality of first fraction sample storage loop connection ports corresponding to the unit fraction section A plurality of second fraction sample storage loops, each of which includes a first connection bar for connecting one of the plurality of fraction sample storage loops to each other; A connection port; provided adjacent to the plurality of second fraction sample storage loop connection ports, the other end of any one of the plurality of fraction sample storage loops is connected, and the fraction sample is discharged. 1 fraction test And a second connection for connecting any one of the first fraction sample discharge port and the plurality of second fraction sample storage loop connection ports so as to communicate with each other corresponding to the unit fraction section. A bar can be included.

  The sample supply module includes a first sample inlet port connected to a sample injector, a first sample outlet port provided adjacent to the first sample inlet port, and a first solvent inlet connected to a first pump. A first solvent discharge port provided adjacent to the first solvent inflow port, and a first sample storage loop connection port and a second sample storage loop connection port to which both ends of the sample storage loop are connected, respectively. A first sample supply valve; a second sample inlet port connected to the first solvent outlet port; and a second sample inlet port adjacent to the second sample inlet port and connected to the first fraction sample inlet port. Both ends of the second sample discharge port, the second sample inflow port, and the second sample discharge port are connected to each other, and the sample is separated into the second sample discharge port. It may include a second sample supply valve with an arm.

  The column selection module is connected to the first fraction sample discharge port and provides a path for alternately supplying the plurality of fraction samples to the first solid phase extraction column or the second solid phase extraction column; A column equilibration valve for alternately equilibrating the first solid phase extraction column and the first reverse phase liquid chromatography column or the second solid phase extraction column and the second reverse phase liquid chromatography column; and the column equilibrium And a plurality of fractionated samples are received and alternately supplied to the first solid phase extraction column or the second solid phase extraction column, and the first reversed-phase liquid is supplied from the first solid phase extraction column. The fraction sample is eluted into the chromatography column or the fraction sample is eluted from the second solid phase extraction column into the second reverse phase liquid chromatography column. It is possible to include a non-selection valve.

  The column equilibration valve is connected to the first fraction sample discharge port and a second pump for supplying a second solvent to a second pump for supplying a second solvent; a third pump for supplying a third solvent; A third solvent inflow port through which the third solvent flows; provided adjacent to the second fraction sample inflow port and selectively connected to the second fraction sample inflow port and the third solvent inflow port; A second fraction sample outlet port; and a third solvent outlet port provided adjacent to the third solvent inlet port and selectively connected to the second fraction sample inlet port and the third solvent inlet port. The fraction sample into which the second solvent is injected in a state where the second fraction sample inflow port and the third solvent discharge port are connected to each other through the column selection valve. Second solid phase extraction column And the third solvent flows through the column selection valve while the third solvent inflow port and the third solvent discharge port are connected to each other, the first solid phase extraction column and the first reverse phase liquid chromatography column. Alternatively, the second solid phase extraction column and the second reverse phase liquid chromatography column can be equilibrated alternately.

  The column selection valve is connected to the third solvent discharge port, and is connected to the fraction sample or a fraction sample into which the third solvent flows and a third solvent inflow port; a fourth pump for supplying a fourth solvent. A fourth solvent inflow port through which the fourth solvent flows; provided adjacent to the fraction sample and the third solvent inflow port, and selected as the fraction sample, the third solvent inflow port, and the fourth solvent inflow port. A fractionated sample and a third solvent outlet port connected to each other; and the fourth solvent inflow port adjacent to the fractionated sample, the third solvent inflow port, and the fourth solvent inflow port selectively. A fractionation sample and a third solvent inflow port, the fractionation sample and a third solvent discharge port are connected, and the fractionation sample is supplied to the first solid phase extraction column. After The fourth solvent elutes the fraction sample from the first solid phase extraction column to the first reverse phase liquid chromatography column in a state where the four solvent inflow port is connected to the fraction sample and the third solvent discharge port. The third solvent equilibrates the second solid phase extraction column and the second reverse phase liquid chromatography column in a state where the fraction sample, the third solvent inflow port and the fourth solvent discharge port are connected. The fraction sample and the third solvent inflow port are connected to the fourth solvent discharge port, and the fraction sample is supplied to the second solid phase extraction column, and then the fourth solvent inflow port and the fourth solvent inflow port. The fourth solvent elutes the fractionated sample from the second solid phase extraction column to the second reverse phase liquid chromatography column with the solvent discharge port connected, and the fractionated sample and the third solvent inflow port. It is possible to balance the said third solvent is the first solid phase extraction column and the first reverse-phase liquid chromatography column in a state where the fraction sample and the third solvent emissions tote is connected.

  The double column valve includes a first solid phase extraction column connection port and a first solid phase extraction column path port respectively connected to both ends of the first solid phase extraction column; the fractionated sample and a third solvent discharge port; A first solid phase extraction column inlet port connected to the first solid phase extraction column connection port and the first solid phase extraction column path port; and connected to the first reverse phase liquid chromatography column; A first reversed-phase liquid chromatography column port connected to or disconnected from the first solid-phase extraction column connection port; a second solid-phase extraction column connection port connected to both ends of the second solid-phase extraction column; And a second solid phase extraction column path port; connected to the fourth solvent discharge port and the second solid phase extraction column connection port and the second solid phase extraction column path port. A second solid phase extraction column inlet port selectively coupled to the second reverse phase liquid chromatography column and a second reverse coupled to or disconnected from the second solid phase extraction column connection port. A phase liquid chromatography column port may be included.

  The dual column valve is provided adjacent to the first solid phase extraction column path port, and is connected to or disconnected from the first solid phase extraction column path port; and the first discharge port; And a second discharge port connected to or disconnected from the second solid phase extraction column path port.

  The embodiment of the present invention is a non-continuous sample fraction in which a plurality of fraction samples are automatically obtained by collectively storing samples supplied during corresponding unit fraction intervals within each unit sample supply time. By providing the integrated device, the complexity of the sample can be reduced, the uniformity of the fraction sample can be increased, the fraction reproducibility can be improved, and the loss of the sample can be prevented.

It is a figure which shows the distribution of the identified peptide, after performing RPLC separation at pH2.6 after performing RPLC separation at pH10 and analyzing using a mass spectrometer. It is a figure for demonstrating the technique which integrates a sample discontinuously after fractionation. It is a figure which shows the acquisition operation | movement of a 1st fraction sample in the dual online multifunction liquid chromatography system by one Example of this invention. It is a figure which shows acquisition operation | movement of a 2nd fraction sample in the dual on-line multifunctional liquid chromatography system by one Example of this invention. It is a figure which shows acquisition operation | movement of a 10th fraction sample in the double online multifunction liquid chromatography system by one Example of this invention. It is a figure which shows the operation | movement which supplies a 1st fraction sample to a 1st solid phase extraction column in the dual online multifunction liquid chromatography system by one Example of this invention. In the dual on-line multifunction liquid chromatography system according to an embodiment of the present invention, the first fraction sample is supplied to the first reverse phase liquid chromatography column, and the second solid phase extraction column and the second reverse phase liquid chromatography are supplied. It is a figure which shows the operation | movement which equilibrates a column. It is a figure which shows the operation | movement which supplies a 2nd fraction sample to a 2nd solid-phase extraction column in the dual online multifunction liquid chromatography system by one Example of this invention. In the dual on-line multifunction liquid chromatography system according to an embodiment of the present invention, the second fraction sample is supplied to the second reverse phase liquid chromatography column, and the first solid phase extraction column and the first reverse phase liquid chromatography are supplied. It is a figure which shows the operation | movement which equilibrates a column.

  For a full understanding of the invention and the advantages of the operations and of the objects attained by the practice of the invention, reference is made to the accompanying drawings illustrating the preferred embodiments of the invention and the contents described in the accompanying drawings. The

  Hereinafter, the present invention will be described in detail by explaining preferred embodiments of the present invention with reference to the accompanying drawings. The same reference numerals shown in the drawings indicate the same members.

  FIG. 3 is a diagram showing an operation of acquiring a first fraction sample in a dual online multifunction liquid chromatography system according to an embodiment of the present invention, and FIG. 4 is a dual online multifunction liquid chromatography according to an embodiment of the present invention. FIG. 5 is a diagram illustrating an operation of acquiring a second fraction sample in the graphy system, and FIG. 5 is a diagram illustrating an operation of acquiring the tenth fraction sample in the dual online multifunction liquid chromatography system according to an embodiment of the present invention. 6 is a diagram showing an operation of supplying a first fraction sample to a first solid phase extraction column in a dual online multifunction liquid chromatography system according to an embodiment of the present invention, and FIG. 7 is an embodiment of the present invention. In the dual on-line multifunction liquid chromatography system, the first fraction sample is supplied to the first reversed-phase liquid chromatography column and the second solid-phase extraction is performed. FIG. 8 is a diagram showing an operation of equilibrating the ram and the second reversed-phase liquid chromatography column. FIG. FIG. 9 is a diagram illustrating an operation of supplying an extraction column. FIG. 9 illustrates a dual online multifunction liquid chromatography system according to an embodiment of the present invention, in which a second fraction sample is supplied to a second reverse phase liquid chromatography column. It is a figure which shows the operation | movement which equilibrates 1 solid phase extraction column and a 1st reverse phase liquid chromatography column.

  3 to 5, a dual online multifunction liquid chromatography system 100 according to an embodiment of the present invention continuously receives a sample to be analyzed and obtains and supplies a plurality of fraction samples. And the first reversed-phase liquid chromatography column COL2 are connected to the first reversed-phase liquid chromatography column COL1 and the first reversed-phase liquid chromatography column COL2 is connected to the first reversed-phase liquid chromatography column COL1. Double column valve 400 with second solid phase extraction column SPE2 connected to solid phase extraction column SPE1 and second reverse phase liquid chromatography column COL2, discontinuous sample fraction integration device 200 and double column valve 400, and a plurality of fraction samples sequentially supplied from the discontinuous sample fraction integration device 200 are alternately supplied to the first solid phase. Exits including column SPE1 and the first reverse-phase liquid chromatography column COL1 or second solid phase extraction column SPE2 and column selection module 300 supplies the second reverse-phase liquid chromatography column COL2.

  3 to 5, the discontinuous sample fraction integration device 200 according to the present embodiment serves to acquire a plurality of fraction samples from a sample continuously received from the sample injector S.

  Hereinafter, a plurality of fraction samples according to this example will be described.

  First, a unit sample supply time is set by dividing the total sample supply time for receiving a sample into a plurality of equal parts. Then, a unit fraction section in which each of the plurality of unit sample supply times is equally divided into a plurality is set. A plurality of fraction samples are obtained by integrating and storing samples received during corresponding unit fraction intervals within each unit sample supply time.

  For example, when the total sample supply time is 100 minutes, the unit sample supply times obtained by equally dividing the total sample supply time into five are 20-minute intervals. That is, the first unit sample supply time is 0 to 20 minutes, and the second to fifth unit sample supply times correspond to 20 to 40 minutes, 40 to 60 minutes, 60 to 80 minutes, and 80 to 100 minutes, respectively.

  The first to fifth unit sample supply times may have unit fraction sections that are equally divided into ten. For example, during the first unit sample space time, the first unit fraction interval is 0 to 2 minutes, and the second to tenth unit fraction intervals are 2 to 4 minutes, 4 to 6 minutes, and 6 to 8 minutes, respectively. 8-10 minutes, 10-12 minutes, 12-14 minutes, 14-16 minutes, 16-18 minutes, 18-20 minutes. The first unit fraction interval is 20 to 22 minutes during the second unit sample space time, and the second to tenth unit fraction intervals are 22 to 24 minutes, 24 to 26 minutes, and 26 to 28 minutes, respectively. 28 to 30 minutes, 30 to 32 minutes, 32 to 34 minutes, 34 to 36 minutes, 36 to 38 minutes, and 38 to 40 minutes.

  In this embodiment, samples received during corresponding first to tenth unit fraction intervals within the first to fifth unit sample supply times are stored in an integrated manner to obtain a plurality of fractionated samples. For example, the first unit fraction section within the first unit sample supply time to the fifth unit sample supply time (i.e., 0-2 minutes, 20-22 minutes, 40-42 minutes, 60-62 minutes, 80-82 minutes). ) Is integratedly stored to obtain a first fraction sample. Since the second fraction sample to the tenth fraction sample are similar to the process of obtaining the first fraction sample, detailed description thereof is omitted.

  In this example, the total sample supply time was set to 100 minutes, the first to fifth unit sample supply times were set, and each unit sample supply time was set to 10 unit fraction intervals. However, the scope of rights of the present invention is not limited to this, and various settings can be made for the total sample supply time, the unit sample supply time, and the unit fraction section.

  The discontinuous sample fraction integration device 200 according to the present embodiment is connected to a sample supply module 210 that supplies a sample, and the sample supply module 210, and receives a sample continuously from the sample supply module 210, and a plurality of fraction samples. And a sample fractionation module 250 that sequentially supplies a plurality of fractionation samples to the column selection module 300.

  The sample supply module 210 according to the present embodiment serves to supply samples to the sample fractionation module 250 continuously.

  The sample supply module 210 is connected to the first sample supply valve 220 to which the first pump P 1 for supplying the first solvent and the sample injector S are connected, and the first sample supply valve 220. And a second sample supply valve 240 that receives the sample and supplies it to the sample fractionation module 250.

  The first sample supply valve 220 receives the sample from the sample injector S and stores the sample in the sample storage loop 221, and then receives the first solvent from the first pump P <b> 1 and converts the sample stored in the sample storage loop 221 into the second sample. The supply valve 240 is supplied.

  For this purpose, the first sample supply valve 220 includes a first sample inlet port 222 connected to the sample injector S, a first sample outlet port 223 provided adjacent to the first sample inlet port 222, and a first A first solvent inlet port 226 connected to the pump P1, a first solvent outlet port 227 provided adjacent to the first solvent inlet port 226 and connected to the second sample supply valve 240, and a sample storage loop 221; A first sample storage loop connection port 224 and a second sample storage loop connection port 225 are connected to both ends.

  Although not shown, a first sample storage loop connection port 224 and a second sample storage loop connection port 225 connected to both ends of the sample storage loop 221 are connected to the first sample inflow port 222 and the first sample discharge port 223, respectively. In a connected state, the sample is supplied to the first sample inlet port 222 through the sample injector S and continuously stored in the sample storage loop 221.

  The first sample storage loop connection port 224 and the second sample storage loop connection port 225 are connected to the first solvent inflow port 226 and the first solvent discharge port 227, respectively, and the first solvent inflow port through the first pump P1. The first solvent is injected into 226, and the sample stored in the sample storage loop 221 is continuously supplied to the second sample supply valve 240. At this time, the first pump P1 is composed of a 99% A solution which is a 10 mM triethylammonium bicarbonate (TEAB) in water and a 10 mM triethylammonium bicarbonate acetonitrile solution (10 mM triethylammonium bicarbonate (TEAB) in acid). A first solvent having a pH of 7.5 is supplied to the first solvent inflow port 226.

  In addition, the second sample supply valve 240 continuously injects the sample supplied from the first sample supply valve 220 into the sample separation column 243, and continues the sample injected into the sample separation column 243 into the sample fractionation module 250. The role of supplying it is performed.

  The second sample supply valve 240 is provided adjacent to the second sample inflow port 241 connected to the first solvent discharge port 227 and the second sample inflow port 241, and is connected to the first fraction sample inflow port 261. A second sample discharge port 242, a sample separation column 243 that is connected to the second sample discharge port 241 and the second sample discharge port 242, and separates and elutes the sample at the second sample discharge port 242.

  The sample stored in the sample storage loop 221 is injected into the sample separation column 243 through the first solvent discharge port 227 and the second sample inflow port 241 by the first solvent. The sample injected into the sample separation column 243 is separated and eluted by the first solvent supplied from the first pump P1, and then supplied to the sample fractionation module 250 described later along the second sample discharge port 242. . At this time, the first pump P1 has a solution of 10 mM Triethylammonium bicarbonate (TEAB) in water reduced from 99% to 50%, and a 10 mM Triethylammonium bicarbonate solution (10 mM Triethylammonium bicarbonate) (10 mM Triethylammonium bicarbonate) TEAB) A first solvent, which is a mixed solution in which the B solution, which is in acetone, increases from 1% to 50%, is supplied to the first solvent inflow port 226. Thus, the concentration of the B solution of the first solvent is gradually increased so that the sample is easily eluted from the sample separation column 243.

  The sample fraction module 250 according to the present embodiment serves to acquire ten fraction samples from the sample continuously supplied from the sample supply module 210.

  The sample fraction module 250 is connected to the sample supply module 210, and includes a first fraction valve 260 into which a sample flows, a second fraction valve 280 provided adjacent to the first fraction valve 260, and one end at a first end. A plurality of fraction sample storage loops 284a to 284j connected to the first fraction valve 260 and having the other end connected to the second fraction valve 280;

  In this embodiment, each unit sample supply time has 10 unit fraction intervals, so that the fraction sample storage loops 284a to 284j correspond to the number of corresponding unit fraction intervals in the unit sample supply time. 10 are provided.

  The first fraction valve 260 is provided adjacent to the first fraction sample inflow port 261 through which the sample flows from the second sample discharge port 242 and the first fraction sample inflow port 261. A plurality of first fraction sample storage loop connection ports 262 respectively connected to one ends of the sample storage loops 284a to 284j, a first fraction sample inflow port 261 and a plurality of first fractions corresponding to the unit fraction section. And a first connection bar 263 for connecting any one of the sample storage loop connection ports 262 so as to communicate with each other. In this embodiment, ten first fraction sample storage loop connection ports 262 are provided corresponding to the number of ten fraction sample storage loops 284a to 284j.

  The second fraction valve 280 includes a plurality of second fraction sample storage loop connection ports 282 to which the other ends of the plurality of fraction sample storage loops 284a to 284j are respectively connected, and a plurality of second fraction sample storage ports. A first fraction sample discharge port 281 provided adjacent to the loop connection port 282 and connected to the other end of any one of the plurality of fraction sample storage loops 284a to 284j; Corresponding to the fractionation section, a first fraction sample discharge port 281 and a second connection bar 283 for connecting any one of the plurality of second fraction sample storage loop connection ports 282 so as to communicate with each other are included. In this embodiment, ten second fraction sample storage loop connection ports 282 are provided corresponding to the number of ten fraction sample storage loops 284a to 284j.

  First, a process of acquiring the first to tenth fraction samples in the first unit fraction section to the tenth unit fraction section within the first unit sample supply time (0 to 20 minutes) will be described as follows. .

  As shown in FIG. 3, the operation of acquiring the first fraction sample in the first unit fraction section (0 to 2 minutes) is as follows.

  The first connection bar 263 is connected to the first fraction sample storage loop connection port 262 connected to one end of the first fraction sample storage loop 284a among the plurality of first fraction sample storage loop connection ports 262. The connection bar 283 is connected to a second fraction sample storage loop connection port 282 connected to the other end of the first fraction sample storage loop 284a among the plurality of second fraction sample storage loop connection ports 282.

  Then, in a state where the first solvent discharge port 227 and the second sample inflow port 241 are connected, the first solvent is supplied from the first pump P1 to the sample separation column 243, and the sample is separated and eluted by the sample separation column 243. The sample separated and eluted by the separation column 243 is supplied to the first fraction sample storage loop 284a through the second sample discharge port 242 and the first fraction sample inflow port 261 for storage.

  Moreover, as shown in FIG. 4, the operation | movement which acquires a 2nd fraction sample in a 2nd unit fraction area (2-4 minutes) is as follows.

  The first connection bar 263 is connected to the first fraction sample storage loop connection port 262 connected to one end of the second fraction sample storage loop 284b among the plurality of first fraction sample storage loop connection ports 262. The connection bar 283 is connected to a second fraction sample storage loop connection port 282 connected to the other end of the second fraction sample storage loop 284b among the plurality of second fraction sample storage loop connection ports 282.

  Then, in a state where the first solvent discharge port 227 and the second sample inflow port 241 are connected, the first solvent is supplied from the first pump P1 to the sample separation column 243, and the sample is separated and eluted by the sample separation column 243. The sample separated and eluted by the separation column 243 is supplied to the second fraction sample storage loop 284b through the second fraction discharge port 242, the first fraction sample inflow port 261, and stored.

  Moreover, as shown in FIG. 5, the operation | movement which acquires a 10th fraction sample in a 10th unit fraction area (18-20 minutes) is as follows.

  The first connection bar 263 is connected to the first fraction sample storage loop connection port 262 connected to one end of the tenth fraction sample storage loop 284j among the plurality of first fraction sample storage loop connection ports 262. The connection bar 283 is connected to the second fraction sample storage loop connection port 282 connected to the other end of the tenth fraction sample storage loop 284j among the plurality of second fraction sample storage loop connection ports 282.

  Then, in a state where the first solvent discharge port 227 and the second sample inflow port 241 are connected, the first solvent is supplied from the first pump P1 to the sample separation column 243, and the sample is separated and eluted by the sample separation column 243. The sample separated and eluted by the separation column 243 is supplied to the tenth fraction sample storage loop 284j via the first fraction sample inflow port 261 through the second sample discharge port 242, and stored.

  The first unit sample supply time (0 to 20 minutes) described above with respect to the second to fifth unit sample supply times (20 to 40 minutes, 40 to 60 minutes, 60 to 80 minutes, and 80 to 100 minutes), respectively. The first to tenth fraction samples can be obtained by repeating the process of obtaining the first to tenth fraction samples in the first unit fraction section to the tenth unit fraction section. For example, the first unit fraction section within the first unit sample supply time to the fifth unit sample supply time (i.e., 0-2 minutes, 20-22 minutes, 40-42 minutes, 60-62 minutes, 80-82 minutes). ) Is integratedly stored to obtain a first fraction sample.

  On the other hand, in order to separate and analyze the first to tenth fraction samples obtained by the sample fraction module 250 described above, the first to tenth fraction samples are used as the first reverse phase liquid chromatography column COL1 or the second reverse phase. Supply to the liquid chromatography column COL2.

  For this purpose, the column selection module 300 according to the present embodiment alternately replaces the first to tenth fraction samples sequentially supplied from the sample fraction module 250 with the first fixed column provided in the double column valve 400 described later. It performs the role of supplying to the phase extraction column SPE1 and the first reverse phase liquid chromatography column COL1 or the second solid phase extraction column SPE2 and the second reverse phase liquid chromatography column COL2.

  The column selection module 300 also converts the first to tenth fraction samples into the first solid phase extraction column SPE1 and the first reverse phase liquid chromatography column COL1 or the second solid phase extraction column SPE2 and the second reverse phase liquid chromatography. The first reversed phase liquid chromatography column COL1 or the second solid phase extraction column SPE2 and the second reversed phase liquid chromatography column COL2 are supplied to any one of the columns COL2 and the separation analysis process for the fractionated sample is performed. It is responsible for performing the balancing process for the remaining one at the same time.

  The column selection module 300 is connected to the first fraction sample discharge port 281 and provides a path for alternately supplying a plurality of fraction samples to the first solid phase extraction column SPE1 or the second solid phase extraction column SPE2. A column equilibration valve 310 that alternately equilibrates the first solid phase extraction column SPE1 and the first reverse phase liquid chromatography column COL1 or the second solid phase extraction column SPE2 and the second reverse phase liquid chromatography column COL2, It is connected to the column equilibration valve 310, receives a plurality of fraction samples, and supplies them alternately to the first solid phase extraction column SPE1 or the second solid phase extraction column SPE2, and from the first solid phase extraction column SPE1 to the first reverse phase The fractionated sample is separated and eluted in the liquid chromatography column COL1 or the second reversed phase liquid chromatography from the second solid phase extraction column SPE2. And a column selection valve 330 column COL2 binary image sample is to be separated eluted.

  The column equilibration valve 310 according to the present embodiment serves to provide a path through which the first to tenth fraction samples are alternately supplied to the first solid phase extraction column SPE1 or the second solid phase extraction column SPE2.

  The column equilibration valve 310 includes a second fraction sample inflow port 311 connected to the first fraction sample discharge port 281 and the second pump P2 that supplies the second solvent, and a third pump P3 that supplies the third solvent. And is provided adjacent to the second fraction sample inflow port 311 and the second fraction sample inflow port 311, and is selected as the second fraction sample inflow port 311 and the third solvent inflow port 313. The second fraction sample discharge port 312 and the third solvent inflow port 313 are provided adjacent to each other, and are selectively connected to the second fraction sample inflow port 311 and the third solvent inflow port 313. A third solvent discharge port 334.

  Referring to FIG. 6, the second fraction sample inlet port 311 is connected to the first fraction sample outlet port 281, and the second fraction sample inlet port 311 is connected to the third solvent outlet port 334. The first fraction sample stored in the one-fraction sample storage loop 284a is supplied to a first solid phase extraction column SPE1 of a double selection valve described later. Further, referring to FIG. 8, the second fraction sample inflow port 311 is connected to the first fraction sample discharge port 281, and the second fraction sample inflow port 311 is connected to the third solvent discharge port 334. Thus, the second fraction sample stored in the second fraction sample storage loop 284b is supplied to the second solid phase extraction column SPE2 of the double selection valve described later.

  As described above, the first to tenth fraction samples stored in the first to tenth fraction sample storage loops 284a to 284j are the first fraction sample discharge port 281, the second fraction sample inflow port 311 and the first fraction sample inflow port 311. The three solid-state extraction columns SPE1 and the second solid-phase extraction column SPE2 can be alternately supplied while being connected to the three-solvent discharge port 334.

  At this time, the first to tenth fraction samples discharged from the first to tenth fraction sample storage loops 284a to 284j are lines connecting the first fraction sample discharge port 281 and the second fraction sample inflow port 311. The second solvent is received through the second pump P2 disposed above. Here, the 2nd pump P2 supplies the 2nd solvent which is 0.2% trifluoroacetic acid aqueous solution (0.2% Trifluoroacetic acid (TFA) in water). The first pump P1 is a 10% triethylammonium bicarbonate (TEAB) in water A solution 99% and a 10 mM triethylammonium bicarbonate acetonitrile solution (10 mM Triethylammonium bicarbonate (TEAB) inleconate in acetate). A first solvent, which is a mixed solution of 1% solution, is supplied to the first solvent inflow port 226. Further, the ratio of the flow rates of the first solvent supplied from the first pump P1 and the second solvent supplied from the second pump P2 may be 1: 9.

  This is because the second solvent is injected into the first to tenth fraction samples supplied to the first solid phase extraction column SPE1 or the second solid phase extraction column SPE2 to dilute the first to tenth fraction samples. This is because acidification prevents the first to tenth fraction samples from being lost in the first solid phase extraction column SPE1 or the second solid phase extraction column SPE2 and lowers the composition of the organic solvent.

  Referring to FIG. 7, the third solvent inlet port 313 and the third solvent outlet port 334 are connected while the separation and analysis process is performed on the first fraction sample stored in the first reversed-phase liquid chromatography column COL1. In this state, the third solvent equilibrates the second solid phase extraction column SPE2 and the second reverse phase liquid chromatography column COL2 through the column selection valve 330. In addition, referring to FIG. 9, the third solvent inflow port 313 and the third solvent outlet port 334 are connected while the separation and analysis process is performed on the second fraction sample stored in the second reverse phase liquid chromatography column COL2. In this state, the third solvent equilibrates the first solid phase extraction column SPE1 and the first reverse phase liquid chromatography column COL1 through the column selection valve 330.

  In the present example, the third solvent is a 0.1% formic acid aqueous solution (0.1% Formic acid in water), but is not limited thereto, and the first solid phase extraction column SPE1 and the first reverse phase are not limited thereto. Any aqueous solution that can equilibrate the liquid chromatography column COL1 and the second solid phase extraction column SPE2 and the second reverse phase liquid chromatography column COL2 can be used.

  The column selection valve 330 according to this embodiment receives a plurality of fraction samples from the column equilibration valve 310 and alternately supplies the plurality of fraction samples to the first solid phase extraction column SPE1 or the second solid phase extraction column SPE2. Do the role. The column selection valve 330 supplies the fourth solvent to the first solid phase extraction column SPE1 so that the fractionated sample is eluted from the first solid phase extraction column SPE1 to the first reverse phase liquid chromatography column COL1. Or supplied to the second solid phase extraction column SPE2 so that the fractionated sample is eluted from the second solid phase extraction column SPE2 to the second reverse phase liquid chromatography column COL2.

  The column selection valve 330 is connected to the third solvent discharge port 334, and is connected to the fraction sample or the fraction sample into which the third solvent flows and the third solvent inflow port 331, and the fourth pump P4 that supplies the fourth solvent. The fourth solvent inflow port 333 into which the fourth solvent flows and the fraction sample and the third solvent inflow port 331 are provided adjacent to each other, and the fraction sample and the third solvent inflow port 331 and the fourth solvent inflow port 333 are provided. The fraction sample and the third solvent discharge port 332 and the fourth solvent inflow port 333 are provided adjacent to the fraction sample and the third solvent inflow port 331 and the fourth solvent inflow port 333. And a fourth solvent discharge port 334 that is selectively connected.

  The operation of equilibrating the second solid phase extraction column SPE2 and the second reverse phase liquid chromatography column COL2 will be described. As shown in FIG. 6, the fraction sample, the third solvent discharge port 332, the fraction sample, and the third The first fraction sample is supplied to the first solid phase extraction column SPE1 in a state where the solvent inflow port 331, the third solvent exhaust port 334, and the second fraction sample inflow port 311 are connected. Then, as shown in FIG. 7, in a state where the fourth solvent inflow port 333 is connected to the fraction sample and the third solvent discharge port 332, the fourth solvent removes the first fraction sample from the first solid phase extraction column SPE 1. The third solvent is eluted into the first reversed-phase liquid chromatography column COL1 and the third solvent is in the second solid state with the fraction sample and the third solvent inflow port 331, the fourth solvent exhaust port 334, and the third solvent exhaust port 334 connected. It flows into the phase extraction column SPE2 and the second reverse phase liquid chromatography column COL2 to equilibrate the second solid phase extraction column SPE2 and the second reverse phase liquid chromatography column COL2.

  In the present example, the fourth pump P4 is a 0.1% formic acid aqueous solution (C solution) that is reduced from 99% to 60%, and a 0.1% formic acid acetonitrile solution (0. The fourth solvent, which is a mixed solution in which the D solution, which is 1% Formic acid in actitrile) increases from 1% to 40%, is supplied to the fourth solvent inflow port 333. As described above, the concentration of the D solution of the fourth solvent is gradually increased so that the first fraction sample can be easily eluted by the first solid phase extraction column SPE1, or the second solid phase extraction column SPE2 The second fraction sample is easily eluted.

  Also, the operation of equilibrating the first solid phase extraction column SPE1 and the first reverse phase liquid chromatography column COL1 will be described. As shown in FIG. 8, the fourth solvent discharge port 334, the fractionated sample, and the third solvent inflow After the second fraction sample is supplied to the second solid phase extraction column SPE2 in a state where the port 331, the third solvent discharge port 334, and the second fraction sample inflow port 311 are connected, as shown in FIG. In a state where the fourth solvent inflow port 333 and the fourth solvent discharge port 334 are connected, the fourth solvent elutes the second fraction sample from the second solid phase extraction column SPE2 to the second reverse phase liquid chromatography column COL2, In the state where the fraction sample and the third solvent inflow port 331 and the fraction sample and the third solvent discharge port 332 are connected, the third solvent is the first solid phase extraction column SPE1 and the first reverse phase liquid chromatography. Equilibrating a first solid phase extraction column SPE1 the first reverse-phase liquid chromatography column COL1 flows into fee column COL1.

  On the other hand, the double column valve 400 according to the present embodiment may be used while the separation analysis for the fraction sample is performed in any one of the first reversed phase liquid chromatography column COL1 and the second reversed phase liquid chromatography column COL2. It serves to equilibrate to the other column.

  The double column valve 400 includes a first solid phase extraction column SPE1 connected to the first reverse phase liquid chromatography column COL1 and a second solid phase extraction column SPE2 connected to the second reverse phase liquid chromatography column COL2. . The fraction sample is supplied to the first reverse phase liquid chromatography column COL1 through the first solid phase extraction column SPE1 or to the second reverse phase liquid chromatography column COL2 through the second solid phase extraction column SPE2.

  The double column valve 400 supplies the first to tenth fraction samples alternately to the first reversed phase liquid chromatography column COL1 or the second reversed phase liquid chromatography column COL2, and the first reversed phase. A column selection valve 330 that allows equilibration with respect to the other column at the same time as the separation analysis for the fractionated sample proceeds in either one of the liquid chromatography column COL1 or the second reverse phase liquid chromatography column COL2. In fluid communication.

  For this purpose, the double column valve 400 includes a first solid phase extraction column connection port 412 and a first solid phase extraction column path port 413 connected to both ends of the first solid phase extraction column SPE1, respectively, A first solid phase extraction column inflow port 411 connected to the third solvent discharge port 332 and selectively connected to the first solid phase extraction column connection port 412 and the first solid phase extraction column path port 413; The first reverse phase liquid chromatography column port 414 connected to the reverse phase liquid chromatography column COL1 and connected to or disconnected from the first solid phase extraction column connection port 412 and the both ends of the second solid phase extraction column SPE2 respectively. The second solid phase extraction column connection port 432 and the second solid phase extraction column path port 433 to be connected, and the fourth solvent discharge port 33 are connected. And a second solid phase extraction column inflow port 431 selectively connected to the second solid phase extraction column connection port 432 and the second solid phase extraction column path port 433, and a second reverse phase liquid chromatography column A second reversed phase liquid chromatography column port 434 connected to COL2 and connected to or disconnected from the second solid phase extraction column connection port 432.

  With reference to FIG. 6, the operation of supplying the first fraction sample to the first solid phase extraction column SPE1 will be described. The first solid phase extraction column connection port 412, the first solid phase extraction column inflow port 411, the fraction sample and third solvent discharge port 332, the fraction sample and third solvent inflow port 331, and the third solvent discharge port 334 are connected. In this state, the first fraction sample is supplied to the first solid phase extraction column SPE1. The double column valve 400 further includes a first discharge port 451 provided adjacent to the first solid phase extraction column path port 413 and connected to or disconnected from the first solid phase extraction column path port 413. When the first fraction sample is supplied to the first solid phase extraction column SPE1 and concentrated, the salt is discharged to the outside through the first discharge port 451.

  Referring to FIG. 7, the first reverse phase liquid chromatography column port 414 is used to inject the first fraction sample into the first reverse phase liquid chromatography column COL1 and proceed with the separation and analysis of the first fraction sample. In the state where the first solid phase extraction column connection port 412, the first solid phase extraction column path port 413, and the first solid phase extraction column inflow port 411 are connected, the fourth solvent becomes the fourth solvent inflow port 333 and the fractionated sample. And flows into the first solid phase extraction column SPE1 along the third solvent discharge port 332 and elutes the first fraction sample by the first solid phase extraction column SPE1, and the first fraction sample is subjected to the first reverse phase liquid chromatography. Supply to the chromatography column COL1. In addition, in order to equilibrate the second solid phase extraction column SPE2 and the second reverse phase liquid chromatography column COL2 simultaneously with the separation analysis for the first fraction sample, the second reverse phase liquid chromatography column port 434 and the second solid phase In a state where the extraction column connection port 432, the second solid phase extraction column path port 433, and the second solid phase extraction column inflow port 431 are connected, the third solvent is a fraction sample, the third solvent inflow port 331, and the fourth solvent discharge. The gas flows into the second solid phase extraction column SPE2 and the second reverse phase liquid chromatography column COL2 along the port 334 to equilibrate the second solid phase extraction column SPE2 and the second reverse phase liquid chromatography column COL2.

  In addition, an operation of supplying the second fraction sample to the second solid phase extraction column SPE2 will be described with reference to FIG. In a state where the second solid phase extraction column connection port 432, the second solid phase extraction column inflow port 431, the fourth solvent discharge port 334, the fractionated sample, and the third solvent inflow port 331 and the third solvent discharge port 334 are connected. The second fraction sample is supplied to the second solid phase extraction column SPE2. The double column valve 400 further includes a second discharge port 453 provided adjacent to the second solid phase extraction column path port 433 and connected to or disconnected from the second solid phase extraction column path port 433. When the second fraction sample is supplied to the second solid phase extraction column SPE2 and concentrated, the salt is discharged to the outside through the second discharge port 453.

  Referring to FIG. 9, the second reverse phase liquid chromatography column port 434 is used to inject the second fraction sample into the second reverse phase liquid chromatography column COL2 and proceed with the separation analysis on the second fraction sample. The fourth solvent is connected to the fourth solvent inflow port 333 and the fourth solvent in a state where the second solid phase extraction column connection port 432, the second solid phase extraction column path port 433, and the second solid phase extraction column inflow port 431 are connected. It flows into 2nd solid-phase extraction column SPE2 along the discharge port 334, a 2nd solid-phase extraction column SPE2 elutes a 2nd fraction sample, and a 2nd fraction sample is 2nd reverse phase liquid chromatography column COL2 To supply.

  In addition, in order to equilibrate the first solid phase extraction column SPE1 and the first reverse phase liquid chromatography column COL1 simultaneously with the separation analysis for the second fraction sample, the first reverse phase liquid chromatography column port 414 and the first solid phase In a state where the extraction column connection port 412, the first solid phase extraction column path port 413, and the first solid phase extraction column inflow port 411 are connected, the third solvent is the fraction sample, the third solvent inflow port 331, the fraction sample, and The first solid phase extraction column SPE1 and the first reverse phase liquid chromatography column COL1 flow along the third solvent discharge port 332 to equilibrate the first solid phase extraction column SPE1 and the first reverse phase liquid chromatography column COL1. To do.

  In the above-described manner, the third to tenth fraction samples are alternately applied to the first solid phase extraction column SPE1 and the first reverse phase liquid chromatography column COL1 or the second solid phase extraction column SPE2 and the second reverse phase liquid chromatography. The first solid phase extraction column SPE1 and the first reversed phase liquid chromatography column COL1 or the second solid phase extraction column SPE2 and the second reversed phase liquid chromatography column COL2 are equilibrated while being supplied to the chromatography column COL2. It can be performed.

  As described above, the present invention is not limited to the described embodiments, and various modifications or variations can be made without departing from the spirit and scope of the present invention. It is clear to those who have. Accordingly, it can be said that such modifications or variations belong to the scope of the claims of the present invention.

The present invention automates the discontinuous sample fraction integration process that reduces sample complexity and increases the uniformity of fractionated samples, thereby improving fraction reproducibility and preventing sample loss. .

  The present invention relates to a discontinuous sample fraction integration device and a dual on-line equipped with the samemultidimensionalMore particularly to liquid chromatography systems, and more specifically, a discontinuous sample fraction integration device that reduces sample complexity and increases the uniformity of fractionated samples, and dual online with the same multidimensionalThe present invention relates to a liquid chromatography system.

Therefore, the technical problem to be solved by the present invention is to improve fraction reproducibility by automating the discontinuous sample fraction integration process which reduces the complexity of the sample and increases the uniformity of the fractionated sample. It is to provide a discontinuous sample fraction integration device capable of preventing sample loss and a dual on-line multidimensional liquid chromatography system including the same.

According to another aspect of the present invention, a sample to be analyzed is continuously received, and a unit sample supply time is set by equally dividing a total sample supply time for receiving the sample into a plurality of unit sample supply times. A plurality of fraction samples obtained by integrating and storing the samples received during the corresponding unit fraction intervals within each unit sample supply time A first solid phase in which a first reverse phase liquid chromatography column and a first reverse phase liquid chromatography column are connected and connected to the first reverse phase liquid chromatography column; A dual column valve with a second solid phase extraction column coupled to an extraction column and the second reverse phase liquid chromatography column; and the discontinuous sample fraction integration device and the duplex The fraction samples provided between the ram valves and sequentially supplied from the discontinuous sample fraction integration device are alternately supplied to the first solid phase extraction column and the first reverse phase liquid chromatography column or the second. A dual on-line multidimensional liquid chromatography system can be provided that includes a solid phase extraction column and a column selection module that feeds the second reversed phase liquid chromatography column.

It is a figure which shows the distribution of the identified peptide, after performing RPLC separation at pH2.6 after performing RPLC separation at pH10 and analyzing using a mass spectrometer. It is a figure for demonstrating the technique which integrates a sample discontinuously after fractionation. It is a figure which shows the acquisition operation | movement of a 1st fraction sample in the double online multidimensional liquid chromatography system by one Example of this invention. It is a figure which shows the acquisition operation | movement of a 2nd fraction sample in the double online multidimensional liquid chromatography system by one Example of this invention. It is a figure which shows the acquisition operation | movement of a 10th fraction sample in the double online multidimensional liquid chromatography system by one Example of this invention. It is a figure which shows the operation | movement which supplies a 1st fraction sample to a 1st solid phase extraction column in the double online multidimensional liquid chromatography system by one Example of this invention. In a dual online multidimensional liquid chromatography system according to an embodiment of the present invention, a first fraction sample is supplied to a first reverse phase liquid chromatography column, and a second solid phase extraction column and a second reverse phase liquid chromatography are supplied. It is a figure which shows the operation | movement which equilibrates a column. It is a figure which shows the operation | movement which supplies a 2nd fraction sample to a 2nd solid-phase extraction column in the double online multidimensional liquid chromatography system by one Example of this invention. In a dual online multidimensional liquid chromatography system according to an embodiment of the present invention, a second fraction sample is supplied to a second reverse phase liquid chromatography column, and the first solid phase extraction column and the first reverse phase liquid chromatography are supplied. It is a figure which shows the operation | movement which equilibrates a column.

FIG. 3 is a diagram illustrating an operation of acquiring a first fraction sample in a dual online multidimensional liquid chromatography system according to an embodiment of the present invention, and FIG. 4 is a dual online multidimensional liquid chromatography according to an embodiment of the present invention. FIG. 5 is a diagram showing an operation of acquiring a second fraction sample in the graphy system, and FIG. 5 is a diagram showing an operation of acquiring the tenth fraction sample in the dual online multidimensional liquid chromatography system according to an embodiment of the present invention. 6 is a diagram showing an operation of supplying a first fraction sample to a first solid phase extraction column in a dual online multidimensional liquid chromatography system according to an embodiment of the present invention, and FIG. 7 is an embodiment of the present invention. in dual-line multidimensional liquid chromatography system, it provides a first fraction samples in the first reverse-phase liquid chromatography column, a second solid phase extraction Lamb and illustrates an operation to balance the second reverse-phase liquid chromatography column, in a double-line multidimensional liquid chromatography system according to an embodiment of FIG. 8 is the present invention, the second fraction samples second solid phase FIG. 9 is a diagram illustrating an operation of supplying to an extraction column. FIG. 9 illustrates a dual online multi-dimensional liquid chromatography system according to an embodiment of the present invention. It is a figure which shows the operation | movement which equilibrates 1 solid phase extraction column and a 1st reverse phase liquid chromatography column.

3 to 5, a dual on-line multi-dimensional liquid chromatography system 100 according to an embodiment of the present invention continuously receives a sample to be analyzed and obtains and supplies a plurality of fraction samples. And the first reversed-phase liquid chromatography column COL2 are connected to the first reversed-phase liquid chromatography column COL1 and the first reversed-phase liquid chromatography column COL2 is connected to the first reversed-phase liquid chromatography column COL1. Double column valve 400 with second solid phase extraction column SPE2 connected to solid phase extraction column SPE1 and second reverse phase liquid chromatography column COL2, discontinuous sample fraction integration device 200 and double column valve 400, and a plurality of fraction samples sequentially supplied from the discontinuous sample fraction integration device 200 are alternately supplied to the first solid phase. Exits including column SPE1 and the first reverse-phase liquid chromatography column COL1 or second solid phase extraction column SPE2 and column selection module 300 supplies the second reverse-phase liquid chromatography column COL2.

Claims (16)

A sample supply module for supplying a sample to be analyzed; and a unit sample connected to the sample supply module for continuously receiving the sample and dividing the total sample supply time for receiving the sample from the sample supply module into a plurality of equal parts The sample received between the corresponding unit fraction intervals in each unit sample supply time after setting a supply time and setting a unit fraction interval in which each of the plurality of unit sample supply times is equally divided A non-continuous sample fraction integration device comprising a sample fractionation module for storing a plurality of fractionation samples in an integrated manner. The sample fractionation module includes:
A first fractionation valve connected to the sample supply module and into which the sample flows;
A second fractionation valve provided adjacent to the first fractionation valve; and one end connected to the first fractionation valve and the other end connected to the second fractionation valve and the unit sample supply time A plurality of unit fraction sections corresponding to the number of unit fraction sections within the unit sample supply time, and the samples received during the corresponding unit fraction sections within the unit sample supply time are sequentially and integratedly stored. The discontinuous sample fraction integration device of claim 1, comprising a fraction sample storage loop. The first fraction valve is
A fraction sample inlet port through which the sample flows from the sample supply module;
A plurality of first fraction sample storage loop connection ports provided adjacent to the fraction sample inflow port and respectively connected to one end of the plurality of fraction sample storage loops; and corresponding to the unit fraction section A first connection bar for connecting the fraction sample inflow port and any one of the plurality of first fraction sample storage loop connection ports to communicate with each other;
The second fractionation valve is
A plurality of second fraction sample storage loop connection ports to which the other ends of the plurality of fraction sample storage loops are respectively connected;
Fraction sample discharge that is provided adjacent to the plurality of second fraction sample storage loop connection ports, is connected to one other end of the plurality of fraction sample storage loops, and discharges the fraction sample. And a second connection bar for connecting any one of the fraction sample discharge port and the plurality of second fraction sample storage loop connection ports to communicate with each other in correspondence with the unit fraction section. The discontinuous sample fraction integration device of claim 2. The sample supply module includes:
A first sample supply valve connected to a first pump for supplying a first solvent and a sample injector; and the fractionated sample connected to the first sample supply valve for receiving the sample from the first sample supply valve 4. The discontinuous sample fraction integration device according to claim 3, comprising a second sample supply valve for supplying to the inlet port. The first sample supply valve is
A first sample inlet port coupled to the sample injector;
A first sample outlet port provided adjacent to the first sample inlet port;
A first solvent inlet port connected to the first pump;
A first solvent discharge port provided adjacent to the first solvent inflow port and connected to the second sample supply valve; and a first sample storage loop connection port and a second connected to both ends of the sample storage loop, respectively. Including a sample storage loop connection port;
The sample is stored in the sample storage loop in a state where the first sample storage loop connection port and the second sample storage loop connection port are connected to the first sample inflow port and the first sample discharge port, respectively.
Injecting the first solvent into the sample storage loop with the first sample storage loop connection port and the second sample storage loop connection port connected to the first solvent inflow port and the first solvent discharge port, respectively. The discontinuous sample fraction integration device according to claim 4, wherein the sample is supplied to the second sample supply valve. The second sample supply valve is
A second sample inlet port connected to the first solvent outlet port;
A second sample outlet port provided adjacent to the second sample inlet port and connected to the fractionated sample inlet port; and both ends connected to the second sample inlet port and the second sample outlet port; A sample separation column from which the sample is eluted at the second sample discharge port;
The discontinuous sample fraction integration device according to claim 5, wherein the sample eluted from the sample separation column is supplied to the fraction sample input port. A unit fraction obtained by continuously receiving a sample to be analyzed, setting a unit sample supply time obtained by equally dividing the total sample supply time for receiving the sample into a plurality of units, and dividing each of the plurality of unit sample supply times into a plurality of equal parts. Discontinuous sample fractionation that supplies a plurality of fraction samples obtained by integrated storage of the samples received during corresponding unit fractionation intervals within each unit sample supply time after setting the intervals Integrated device;
A first reversed phase liquid chromatography column and a second reversed phase liquid chromatography column are connected, and a first solid phase extraction column and the second reversed phase liquid chromatography column are connected to the first reversed phase liquid chromatography column. A double column valve with a second solid phase extraction column coupled to the discontinuous sample fraction integration device and the dual column valve, and from the discontinuous sample fraction integration device The sequentially supplied fractionated samples are alternately supplied to the first solid phase extraction column and the first reverse phase liquid chromatography column or to the second solid phase extraction column and the second reverse phase liquid chromatography column. Dual online multifunction liquid chromatography system including column selection module. The discontinuous sample fraction integration device comprises:
A sample supply module for supplying the sample; and connected to the sample supply module, continuously receiving the sample from the sample supply module to obtain a plurality of fraction samples, and supplying the plurality of fractions to the column selection module. The dual on-line multifunction liquid chromatography system according to claim 7, comprising a sample fractionation module for sequentially supplying samples. The sample fractionation module includes:
A first fractionation valve connected to the sample supply module and into which the sample flows;
A second fractionation valve provided adjacent to the first fractionation valve; and one end connected to the first fractionation valve and the other end connected to the second fractionation valve and the unit sample supply time A plurality of unit fraction sections are provided corresponding to the number of unit fraction sections, and the samples received during the corresponding unit fraction sections within each unit sample supply time are sequentially and integratedly stored. The dual on-line multifunction liquid chromatography system of claim 8 comprising a fractional sample storage loop. The first fraction valve is
A first fraction sample inlet port through which the sample flows from the sample supply module;
A plurality of first fraction sample storage loop connection ports provided adjacent to the first fraction sample inflow port and connected to one ends of the plurality of fraction sample storage loops; and corresponding to the unit fraction section A first connection bar connecting the fraction sample inflow port and any one of the plurality of first fraction sample storage loop connection ports so as to communicate with each other;
The second fractionation valve is
A plurality of second fraction sample storage loop connection ports to which the other ends of the plurality of fraction sample storage loops are respectively connected;
A first fraction provided adjacent to the plurality of second fraction sample storage loop connection ports, connected to the other end of any one of the plurality of fraction sample storage loops, and discharging the fraction sample. A sample discharge port; and a second connection for connecting any one of the first fraction sample discharge port and the plurality of second fraction sample storage loop connection ports in communication with each other in correspondence with the unit fraction section. The dual on-line multifunction liquid chromatography system of claim 9 comprising a linking bar. The sample supply module includes:
A first sample inlet port connected to a sample injector; a first sample outlet port provided adjacent to the first sample inlet port; a first solvent inlet port connected to a first pump; A first sample supply valve including a first solvent discharge port provided adjacent to the solvent inflow port, and a first sample storage loop connection port and a second sample storage loop connection port to which both ends of the sample storage loop are respectively connected. A second sample inlet port connected to the first solvent outlet port; a second sample outlet port provided adjacent to the second sample inlet port and connected to the first fraction sample inlet port; A second sample supply comprising a sample separation column having both ends connected to the second sample inlet port and the second sample outlet port, and the sample is eluted to the second sample outlet port. 11. The dual on-line multifunction liquid chromatography system of claim 10, comprising a valve. The column selection module
A path connected to the first fraction sample discharge port and alternately supplying the plurality of fraction samples to the first solid phase extraction column or the second solid phase extraction column; A column equilibration valve for alternately equilibrating a column and the first reverse phase liquid chromatography column or the second solid phase extraction column and the second reverse phase liquid chromatography column; and connected to the column equilibration valve; The plurality of fractionated samples are received and supplied alternately to the first solid phase extraction column or the second solid phase extraction column, and the fractionation from the first solid phase extraction column to the first reverse phase liquid chromatography column. A column selection valve that allows a fraction sample to be eluted or the fraction sample to be eluted from the second solid phase extraction column to the second reverse phase liquid chromatography column. 10. The dual online multifunction liquid chromatography system according to 10. The column equilibration valve is
A second fraction sample inlet port connected to the first fraction sample discharge port and a second pump for supplying a second solvent;
A third solvent inflow port connected to a third pump for supplying a third solvent and into which the third solvent flows;
A second fraction sample outlet port provided adjacent to the second fraction sample inlet port and selectively connected to the second fraction sample inlet port and the third solvent inlet port; and the third solvent A third solvent outlet port provided adjacent to the inlet port and selectively connected to the second fraction sample inlet port and the third solvent inlet port;
The fraction sample into which the second solvent has been injected in a state where the second fraction sample inflow port and the third solvent discharge port are connected to each other through the column selection valve is the first solid phase extraction column or the second fraction extraction sample. Supplied to the solid phase extraction column,
In a state where the third solvent inflow port and the third solvent discharge port are connected, the third solvent passes through the column selection valve and the first solid phase extraction column and the first reverse phase liquid chromatography column or the second solvent. The dual on-line multifunction liquid chromatography system of claim 12, wherein a solid phase extraction column and the second reverse phase liquid chromatography column are equilibrated alternately. The column selection valve is
A fraction sample and a third solvent inflow port connected to the third solvent discharge port and into which the fraction sample or the third solvent flows;
A fourth solvent inflow port connected to a fourth pump for supplying a fourth solvent and into which the fourth solvent flows;
A fraction sample and a third solvent outlet port, which are provided adjacent to the fraction sample and the third solvent inlet port and are selectively connected to the fraction sample, the third solvent inlet port and the fourth solvent inlet port. And a fourth solvent outlet port provided adjacent to the fourth solvent inlet port and selectively connected to the fractionated sample and the third solvent inlet port and the fourth solvent inlet port;
After the fractionated sample and the third solvent inflow port are connected to the fractionated sample and the third solvent outlet port and the fractionated sample is supplied to the first solid phase extraction column, the fourth solvent inflow port and The fourth solvent elutes the fraction sample from the first solid phase extraction column to the first reverse phase liquid chromatography column in a state where the fraction sample and the third solvent discharge port are connected, The third solvent equilibrates the second solid phase extraction column and the second reverse phase liquid chromatography column in a state where the sample and the third solvent inflow port and the fourth solvent discharge port are connected, and the fractionated sample And the third solvent inlet port and the fourth solvent outlet port are connected to supply the fractionated sample to the second solid phase extraction column, and then the fourth solvent inlet port and the fourth solvent outlet port are connected. In the state The fourth solvent elutes the fraction sample from the second solid phase extraction column to the second reverse phase liquid chromatography column, and the fraction sample, the third solvent inflow port, the fraction sample, and the third solvent. 14. The dual on-line multifunction liquid chromatography system of claim 13, wherein the third solvent equilibrates the first solid phase extraction column and the first reverse phase liquid chromatography column with a connected discharge tote. . The double column valve is
A first solid phase extraction column connection port and a first solid phase extraction column path port respectively connected to both ends of the first solid phase extraction column;
A first solid phase extraction column inlet port connected to the fractionated sample and a third solvent discharge port and selectively connected to the first solid phase extraction column connection port and the first solid phase extraction column path port;
A first reverse phase liquid chromatography column port connected to the first reverse phase liquid chromatography column and connected to or disconnected from the first solid phase extraction column connection port;
A second solid phase extraction column connection port and a second solid phase extraction column path port respectively connected to both ends of the second solid phase extraction column;
A second solid phase extraction column inlet port connected to the fourth solvent discharge port and selectively connected to the second solid phase extraction column connection port and the second solid phase extraction column path port; and 15. The dual on-line multifunction liquid of claim 14, comprising a second reverse phase liquid chromatography column port connected to a reverse phase liquid chromatography column and connected or disconnected from the second solid phase extraction column connection port. Chromatography system. The double column valve is
A first discharge port provided adjacent to the first solid phase extraction column path port and connected to or disconnected from the first solid phase extraction column path port; and provided adjacent to the first discharge port; 16. The dual on-line multifunction liquid chromatography system of claim 15, further comprising a second discharge port coupled to or decoupled from the second solid phase extraction column path port.